Gene targeting studies provide compelling evidence that glycolysis in spermatozoa rather than mitochondrial ATP production is essential for maintaining sperm motility and male fertility in the mouse. Although human sperm exhibit high glycolytic activity, there are conflicting reports on the relative importance of glycolysis and mitochondrial oxidative phosphorylation for maintaining ATP levels in these cells. In addition, there are a surprising number of glycolytic variants in mammalian sperm, and recent studies continue to uncover new enzymes and regulatory features of both glycolytic and other metabolic enzymes. Specific aims of this proposal will: i) Determine if energy production in mouse sperm is regulated by novel mechanisms. We will a) determine if substrates other than glucose sustain sperm motility and functional properties sufficient for in vitro fertilization, b) use metabolomic and fluxomic approaches to determine how sperm metabolize effective substrates, and c) determine if PKA-mediated phosphorylation in sperm is essential for maintaining glycolytic ATP production. 2) Identify substrates that maintain ATP production and motility in human sperm and determine how they are metabolized. To test the hypothesis that human sperm require glycolysis for fertilization competence, we will a) determine if both glycolysable and non-glycolysable substrates are required to maintain sperm function, b) use metabolomic approaches to assess how effective substrates are metabolized by human sperm, and c) determine if there is evidence for glycolytic defects in infertile men with low sperm motility. 3) Determine functional characteristics of newly identified glycolytic isozymes that are present in sperm. These studies will test the hypothesis that extensive modifications of sperm glycolysis sperm are essential for alternative regulatory mechanisms and/or binding of pathway constituents to the fibrous sheath to provide a localized supply of ATP along the length of the flagellum. We will a) determine the diversity of LDH isozymes present in mouse sperm, b) determine if aldolase A and novel LDH isozymes present in mouse and human sperm have unique functional properties, and c) use genomic analyses to assess the complexity of isoforms that are used at other steps of the sperm glycolytic pathway. Knowledge gained from these studies will increase our understanding of how glycolysis is regulated in sperm and provide insights regarding the relative utility of specific sperm isozymes as potential contraceptive targets.